The ubiquitin-proteasome system regulates the turnover of proteins that have essential roles in the cell cycle, apoptosis, DMA damage repair, and in protein trafficking, which makes this pathway a target for oncogenic events. Several ubiquitin E3 ligases are proto-oncogenes that degrade tumor suppressor proteins. Breast cancer has an unbalanced proto-oncogenic RING-finger ubiquitin E3 ligase signature. Thus, approaches to restore protein homeostasis could lead to novel breast cancer treatments. We isolated the novel breast cancer associated gene 2 (BCA2) by subtractive hybridization cloning from an invasive breast cell line, and found that it has a RING-finger domain. We demonstrated that the BCA2 RING-finger, a specialized zinc- finger, is responsible for its intrinsic autoubiquitination activity. BCA2 is overexpressed in more than 50% of invasive breast cancers compared to normal tissues. Overexpression of BCA2 increases proliferation of NIH3T3 fibroblasts, whereas small interfering RNA inhibits growth of BCA2-expressing breast cancer cells. BCA2 is expressed in the nucleus and cytoplasm of breast cancer cells suggesting multiple functions. A cytoplasmic binding partner of BCA2 is Rab7, which is involved in receptor endocytosis and recycling. Rab7 was shown to regulate endocytic trafficking of the epidermal growth factor receptor complex. Overexpression of BCA2 leads to inhibition of EGF degradation. In the nucleus, BCA2 appears co-expressed with estrogen receptor. High nuclear BCA2 levels are detected in ER-positive breast cancers, while ER-negative breast cancers have low nuclear BCA2. Moreover, BCA2 is an estrogen responsive gene, suggesting that BCA2 and ER might crosstalk. An inhibitory agent disulfiram, has been identified by us and shows antitumor activity that is related to BCA2 inhibition via ejection of zinc from its catalytic RING domain. Only BCA2-expressing breast cancer cell lines respond to treatment with disulfiram. We hypothesize that BCA2 is a regulator of receptor-mediated signaling pathways that are important in breast cancer and thus that the RING-finger ubiquitin E3 ligase BCA2 is a target for therapeutic intervention. We further hypothesize that ejection of zinc from the RING-finger domain can lead to specific inhibition of its E3 ligase activity. In this proposal we will focus on delineating the cytoplasmic and nuclear functions of BCA2 in breast cancer cells as well as on the structure-based design of BCA2 inhibitory agents. Our Specific Aims are: To use genetic and chemical inhibition for delineating the functional relationships between BCA2 and Rab7, and for studying crosstalk between BCA2 and ER. We will further develop zinc ejecting compounds that can specifically inhibit BCA2 E3 ligase activity. This project could have a major impact on mechanism-based drug discovery for modulation of RING-finger E3 ligase-mediated protein ubiquitination, not only in cancer.